Lipid acyltransferases are known to be advantageous in food applications. Lipid acyltransferases have been found to have significant acyltransferase activity in foodstuffs. This activity has surprising beneficial applications in methods of preparing foodstuffs.
For instance, WO 2004/064537 discloses a method for the in situ production of an emulsifier by use of a lipid acyltransferase and the advantages associated therewith.
Further, lipid:cholesterol acyltransferase enzymes have been known for some time (see for example Buckley—Biochemistry 1983, 22, 5490-5493). In particular, glycerophospholipid:cholesterol acyl transferases (GCATs) have been found, which like the plant and/or mammalian lecithin:cholesterol acyltransferases (LCATs), will catalyse fatty acid transfer between phosphatidylcholine and cholesterol.
Upton and Buckley (TIBS 20, May 1995, p178-179) and Brumlik and Buckley (J. of Bacteriology April 1996, p2060-2064) teach a lipase/acyltransferase from Aeromonas hydrophila which has the ability to carry out acyl transfer to alcohol receptors in aqueous media.
A putative substrate binding domain and active site of the A. hydrophila acyltransferase have been identified (see for example Thornton et al 1988 Biochem. et Biophys. Acta. 959, 153-159 and Hilton & Buckley 1991 J. Biol. Chem. 266, 997-1000) for this enzyme.
Buckley et al (J. Bacteriol 1996, 178(7) 2060-4) taught that Ser16, Asp116 and His291 are essential amino acids which must be retained for enzyme activity to be maintained.
Robertson et al (J. Biol. Chem. 1994, 269, 2146-50) taught some specific mutations, namely Y226F, Y230F, Y30F, F13S, S18G, S18V, of the A. hydrophila acyltransferase, none of which are encompassed by the present invention.
Accordingly, there is a need for a method for the commercial production of lipid acyltransferases, including variant lipid acyltransferases.
However, generally genes can be difficult to express in heterologous hosts and expression of lipid acyltransferases in host cells can be problematic.
WO 2004/064537 discloses the expression of two Aeromonas lipid acyltransferases in Bacillus subtilis and Escherichia Coli. However, expression in B. subtilis is low whilst E. coli is not a GRAS organism and is, therefore, unsuitable as a host for enzymes that are to be used in the food industry.
U.S. Pat. No. 6,255,076 discloses a method of producing a polypeptide in a Bacillus host cell. However, such a method requires the use of a tandem promoter in which each promoter sequence in operably linked to a single copy of a nucleic acid sequence encoding the polypeptide sequence. Thus, there is a need in the art for an improved method for the production of lipid acyltransferases.